The present invention relates to structures involved in the secretory processes of reproductive tissues, including the prostatic secretory processes, and their protein products which may be used as tools for diagnosing reproductive pathology including prostate disease. The present invention also relates to reagents, such as antibodies, other ligands and oligonucleotides, for detecting these structures or their contents and to methods of diagnosing prostate pathology, including prostate cancer and prostatitis. The invention further relates to an improved tissue and cell fixation process for the detection of secretory structures in reproductive tissue. The fixation process is useful for the diagnosis of prostate, testes and renal cancer.
The secretory (luminal) cells of normal prostatic glands are separated from the basement membrane by a layer of inconspicuous basal cells (1). This surface layer is characterised by its tall columnar cells with basally orientated nuclei, whose abundant apical cytoplasm synthesises a broad range of secretory proteases including prostate specific antigen (PSA) and prostatic acid phosphatase (PAP). Characteristically, the cytoplasm of surface secreting cells is optically clear to faintly eosinophilic which distinguishes it dramatically from the amphiphilic (dark) cytoplasmic staining of most dysplastic or malignant prostatic epithelial cells (2,3). Optimal tissue staining for diagnostic purposes yields maximal cytoplasmic clarity in benign prostatic secretory cells (2), the abrupt contrast between the cytoplasmic density of cancer and the pallor of the adjacent normal epithelium in well stained sections is often the most striking histologic feature which delineates the boundaries of a carcinoma focus. Conversely, a frequent problem in needle core diagnosis is the xe2x80x9cclear cellxe2x80x9d glandular atypical focus, in which confirmation of carcinoma is more difficult due to the absence of dark cytoplasm.
The significance of cytoplasmic density in adenocarcinoma is not understood. In the Gleason grading system (4) retention of clear cytoplasm implies a high level of differentiation since it is a requisite feature of all Grade 1 and Grade 2 carcinomas. Furthermore, cytoplasmic clarity is also characteristic of most adenocarcinomas which arise from the transition zone in association with nodular hyperplasia (5,6). Although dark cytoplasm is well described in dysplasia (PIN) and Gleason grade 3 carcinoma, it is not specific for malignant loss of differentiation since it is common in the cells of benign post inflammatory atrophy (7).
The organelles responsible for the appearance of the normal prostatic secretory cell cytoplasm have been described as myriad tiny vesicles which nearly fill the cytoplasm and appear completely devoid of content (1,8). These vesicles or granules are only faintly and variably recognisable by routine light microscopy, depending on the staining intensity of the faintly eosinophilic narrow septa which separate them.
Prostatic corpora amylacea (CA) are extracellular intraluminal structures seen in most adult prostate (9). The protein source of the CA is poorly understood although within these structures a group of sulphur-rich proteins have been previously detected (10). Similar sulphated proteins have also been detected in crystals associated with well-differentiated carcinoma, so-called prostatic crystalloids (11). Further, amyloid possibly related to xcex2-2 microglobulin has been recognized in CA (12) but, despite these basic observations the origins of these extracellular prostatic structures is yet to be determined.
Round proteinaceous deposits have also been identified in prostatic luminae (9). These deposits (3-8 xcexcm in diameter) are rare (8 cases of 166 specimens) and when noted are in close association with the luminal surface of the benign secretory cells confined to the central region of prostate in routinely fixed and processed tissues. The nature of this protein deposit is unknown but was noted to be negative for numerous protein fractions including, prostate specific antigen (PSA), prostatic acid phosphatase (PAP), light chain immunoglobulin (xcexa and xcex), xcex11-antitrypsin and xcex1-fetoprotein (9). Stains for mucin and silver stains were also negative but unexpectedly positive for phloxine tartrazine.
Beyond these initial observations, the detailed structure underlying the xe2x80x9cclear cellxe2x80x9d cytoplasmic appearance of prostatic epithelium as well as the relationships between this appearance and the process of prostatic exocrine secretion have never been systematically studied. Further, common and apparently fundamental alterations of structure and secretory function which must underly the abrupt transition to the dark cytoplasm of most cancers are unknown.
The mammalian oviduct provides an environment that supports the gametes, the process of fertilisation, early embryonic development and the delivery of a viable embryo to the uterus. The lumen of the mammalian oviduct is formed by a complex interdigitating system of longitudinal mucosal folds. These longitudinal mucosal folds are lined by a simple columnar epithelium and the morphological and biochemical characteristics of this epithelium are controlled by ovarian steroids. At the time of ovulation this lining epithelium consists of fully differentiated columnar ciliated and secretory cells. Approximately 40-50% of the epithelial cells are secretory. Secretory glands are observed in the apical regions of the cells. Several secretory products have been identified which enhance sperm motility, viability, binding to zona pellucida and enhance the rate of zona pellucida penetration. (Verhage et al 1997 Characteristics of an oviductal glycoprotein and its potential role in fertility control. J. Reproduction and Fertility Supplement 51:217-226).
The present inventors have now developed improved techniques for visualising prostatic cytoplasmic structure and the mechanism of cell secretion. These improved techniques have led to the surprising finding that secretory granules which are found in the luminal cells of normal prostate glands are absent in prostate carcinomas. These findings indicate that the prostate secretory granules may provide an important tool in the diagnosis of prostate cancers.
The present inventors have also identified a link between PSG, decapitated cytoplasmic body (DCB), eosinophilic bodies (EB) and corpora amylacea (CA) structures. Briefly, the normal secretory cell cytoplasm is filled with brightly eosinophilic PSG measuring about 1 xcexcm in diameter and densely concentrated in the apical third of the cell. This apical compartment represents an apocrine secretory bleb. Periodic detachment of blebs carries packages of secretory granules (PSG) into the lumen where the receptacles fragment liberating their contents. The luminal cytoplasmic compartment (bleb), emptied of its protein enzymes, becomes a decapitated cytoplasmic body (DCB), a partly collapsed, faintly basophilic membrane with remnant cytoplasm. The DCB shrinks to form a sphere with a thickened, brightly eosinophilic surface casing, the eosinophilic body (EB). This structure may dissolve in luminal secretions, but it is also observed that it may adsorb to the surface of the corpus amylaceum (CA).
Unexpectedly, the present inventors have found that in prostatic adenocarcinomas, the entire secretory apparatus is substantially absent; neither PSG, DCB nor EB are found. An inability to form corpora amylacea arises from this fact. Prostate carcinomas may therefore be characterised by a significant reduction in levels, or absence, of any one of the structures associated with prostate secretions.
The present inventors have found that PSGs, EBs, DCBs and CAs may be readily visualised in normal prostate tissue which has been fixed in strong glutaraldehyde, or a substance which produces similar cytoplasmic fixation to that produced by strong glutaraldehyde.
Accordingly, in a first aspect the present invention provides a method of processing a tissue sample for analysis, the method comprising exposing the tissue sample to a composition which produces substantially identical cytoplasmic fixation to that produced by glutaraldehyde at a concentration of at least 2.0% and/or which provides substantially identical preservation of secretory granules as that provided by glutaraldehyde at a concentration of 2.0%.
In a preferred embodiment of the first aspect, the tissue sample is reproductive or renal tissue, more preferably reproductive tissue. Preferably, the reproductive tissue is prostatic, testes, fallopian tube or oviduct tissue.
In a further preferred embodiment, the histological analysis comprises analysing the tissue for the presence of secretory granules.
In a second aspect the present invention provides a method of diagnosing prostate pathology in a subject which method comprises
(i) fixing a sample of prostate tissue from the subject in a fixative which produces substantially identical cytoplasmic fixation to that produced by glutaraldehyde at a concentration of at least 2.5%; and
(ii) analysing the sample for the presence of PSG and/or EB and/or DCB and/or CA structures, or the contents of any one or more of these structures.
It will be appreciated that a reduced number of PSG and/or EB and/or DCB and/or CA structures, or the contents thereof, in the sample is indicative of prostate disease in the subject.
In a preferred embodiment, the method of the second aspect is used to diagnose prostatitis or prostate cancer in the subject.
In one embodiment of the second aspect, the analysis in step (ii) is performed by light microscopy. In this embodiment, the tissue is preferably stained with a stain such as haematoxylin and/or eosin.
In a further embodiment of the second aspect, the analysis in step (ii) involves electron microscopy.
In yet a further embodiment of the second aspect of the present invention, the analysis in step (ii) involves immunostaining. The immunostaining may involve immuno detection of PSA or PAP. Alternatively, the immunostaining may involve immuno detection of PSG and/or EB and/or DCB and/or CA structures.
In a further preferred embodiment of the first and second aspects, the fixative produces substantially identical cytoplasmic fixation to that produced by glutaraldehyde at a concentration of between 2.5% and 6%, more preferably at a concentration of between 3% and 5%. The fixative may comprise glutaraldehyde at a concentration of between 2.5% and 6%, more preferably at a concentration of between 3% and 5%.
In a further preferred embodiment of the first and second aspects, the fixative composition comprises an aqueous solution of glutaraldehyde at a concentration of between 2.5% and 6%, a metallic salt and a buffer stabiliser, the composition having a pH of between 5.7 and 5.75.
In a further preferred embodiment of the first and second aspects, the fixative further comprises phenol, preferably in a concentration ranges of from about 2.0 to about 3.0 g/l, more preferably around 2.5 g/l.
In a further preferred embodiment of the first and second aspects, the metallic salt is selected from the group consisting of zinc sulphate, copper sulphate, barium sulphate, cobalt chloride, barium chloride, potassium chloride, mercuric chloride and lead chloride. Preferably, the metallic salt is zinc sulphate.
In a further preferred embodiment of the first and second aspects, the concentration of the metallic salt ranges from 3.0 to 20.0 g/l, more preferably around 13.0 g/l.
In a further preferred embodiment of the first and second aspects, the buffer comprises one or more acetic acid compounds. Preferably, the buffer stabilizer comprises sodium acetate at a concentration of about 0.2M and acetic acid at a concentration of about 0.2M.
In a further preferred embodiment of the first and second aspects, the fixative further comprises one or more components selected from the group consisting of:
Detergents such as SDS, Tween (0.001%-1.0%),
Azone (laurocaprame 1-dodecylazacyclo-hepton-2-one) 3% w/v or 1-geranylazacyclohepton-2-one 3% w/v,
Liposomes,
Sodium taurocholate 40-0.25 xcexcM; (which may include:
Cholesterol 0.2 mM-0.075mM
Oleicacid 1 mM-0.25mM
Synthetic phospholipids, eg phosphocholin 14-18 10-30 mM as mixed micells),
Solution C24 (polyoxyethene-24-cholesterol-ether),
Polyethylene glycol 200 dilaurate (0.1-10%),
Menthol 1% w/v,
Mercaptoethanol (0.0025%),
Glycerol trioleate,
Terpene penetration enhancers (for example 1,8-cineole, methane, (+)-limonene, nerolidol),
Medium chain fatty acids (caproate C6, C8 caprylate, C10 caprate, C12 laurate),
Trichloroactic acid (0.5-5.0%),
Metallic salts (for example, zinc sulphate, potassium chloride, calcium chloride, zinc chloride) (3-30%),
Dimethylsulfoxide (0.1-20%),
Mono and disaccharides (glucose),
Urea, and
Methyl salicylate.
In a third aspect the present invention provides a histological fixative composition comprising an aqueous solution of glutaraldehyde, a metallic salt and a buffer stabiliser, the composition having a pH of between 5.7 and 5.75.
In a preferred embodiment of the third aspect, the amount of glutaraldehyde ranges from about 2.5% and about 6%, more preferably between about 3.5% and about 5%, by volume of the composition.
In a further preferred embodiment of the third aspect, the fixative comprises phenol, preferably in a concentration ranges of from about 2.0 to about 3.0 g/l, more preferably around 2.5 g/l.
In a further preferred embodiment of the third aspect, the metallic salt is selected from the group consisting of zinc sulphate, copper sulphate, barium sulphate, cobalt chloride, barium chloride, potassium chloride, mercuric chloride and lead chloride. Preferably, the metallic salt is zinc sulphate.
In a further preferred embodiment of the third aspect, the concentration of the metallic salt ranges from 3.0 to 20.0 g/l, more preferably around 13.0 g/l.
In a further preferred embodiment of the third aspect, the buffer comprises one or more acetic acid compounds. Preferably, the buffer stabilizer comprises sodium acetate at a concentration of about 0.2M and acetic acid at a concentration of about 0.2M.
In a further preferred embodiment of the third aspect, the fixative composition further comprises one or more components selected from the group consisting of:
Detergents such as SDS, Tween (0.0.01%-1.0%),
Azone (laurocaprame 1-dodecylazacyclo-hepton-2-one) 3% w/v or 1-geranylazacyclohepton-2-one 3% w/v,
Liposomes,
Sodium taurocholate 40-0.25 xcexcM; (which may include:
Cholesterol 0.2 mM-0.075mM
Oleicacid 1 mM-0.25mM
Synthetic phospholipids, eg phosphocholin 14-18 10-30 mM as mixed micelles),
Solution C24 (polyoxyethene-24-cholesterol-ether),
Polyethylene glycol 200 dilaurate (0.1-10%),
Menthol 1% w/v,
Mercaptoethanol (0.0025%),
Glycerol trioleate,
Terpene penetration enhancers (for example 1,8-cineole, methane, (+)-limonene, nerolidol),
Medium chain fatty acids (caproate C6, C8 caprylate, C10 caprate, C12 laurate),
Trichloroactic acid (0.5-5.0%),
Metallic salts (for example, zinc sulphate, potassium chloride, cobalt chloride, calcium chloride, zinc chloride) (1-30%),
Dimethylsulfoxide (0.1-20%),
Mono and disaccharides (glucose),
Urea, and
Methyl salicylate.
In a particularly preferred embodiment the fixative is prepared as follows:
(i) Phenol (2.5 g) is dissolved in 50 ml of distilled water. The phenol solution is added to 200 ml of glutaraldehyde (25%). The pH of the solution is adjusted to 5.8 by the dropwise addition of 5M NaOH.
(ii) Zinc sulphate (15 g) is dissolved in 250 ml of distilled water. The zinc sulphate solution is then admixed with 470 ml of 0.2 M sodium acetate and 30 ml of 0.2 M acetic acid. The solution is adjusted to a pH of 5.6-5.75.
(iii) The solutions from steps (i) and (ii) are admixed and if necessary, the pH and is adjusted by the addition of NaOH to about 5.7.
Preferably, the final solution is filtered before use.
In a fourth aspect the present invention provides an isolated prostate secretory granule (PSG).
By xe2x80x9cprostate secretory granulexe2x80x9d or xe2x80x9cPSGxe2x80x9d we mean a vesicle which is produced in and secreted from normal prostatic secretory cells.
In a preferred embodiment the PSG has a diameter of 800-1200 nm. In a further preferred embodiment, the PSG has a granular electron dense core without internal membranes.
In a further preferred embodiment, the PSG is eosinophilic.
In a further preferred embodiment, the PSG is glycoprotein and sulphur rich.
In a fifth aspect the present invention provides an isolated prostate cell decapitated cytoplasmic body (DCB).
In a further preferred embodiment, the DCB is glycoprotein and sulphur rich.
In a sixth aspect the present invention provides an isolated prostate eosinophilic body (EB).
In a preferred embodiment, the EB is glycoprotein and sulphur rich.
In a further preferred embodiment, the EB has a diameter of between 4 and 15 xcexcm.
In a seventh aspect the present invention provides an isolated prostate corpora amylacea (CA).
In a preferred embodiment, the CA is glycoprotein and sulphur rich.
The present inventors have found that the contents of the PSG and/or DCB and/or EB and/or CA include sulphur-rich prostatic crystalloids, extracellular acid mucin and sulphate-associated glycosaminoglycans. The sulphate-associated glycosaminoglycans may be rich in glucosamine and galactose. The present inventors have also characterised the sulphated-associated compounds and have identified the substance keratan sulphate as the major sulphur group of PSG, DCB, EB and CA structures.
Accordingly, in an eighth aspect the present invention provides an isolated keratan sulphate-associated compound derived from a PSG and/or DCB and/or EB and/or CA.
One particular keratan sulphate-associated compound has been purified and characterised by molecular weight analysis.
Accordingly, in a ninth aspect the present invention provides a keratan sulphate-associated compound derived from a PSG, the compound having a molecular weight of about 70-75 kDa.
It will be appreciated by those skilled in the art that a PSG and/or DCB and/or EB and/or CA structures or the contents of these structures may be used to generate binding ligands, such as antibodies against a PSG and/or DCB and/or EB and/or CA or the contents thereof. These binding ligands may be used in turn as diagnostic tools in the differentiation of normal and malignant prostate tissue, in situ or by their presence in bodily fluids.
Accordingly, in a tenth aspect the present invention provides a binding ligand, preferably an antibody, directed against a PSG and/or DCB and/or EB and/or CA or the contents thereof.
The term xe2x80x9cantibodyxe2x80x9d is to be construed as covering any specific binding substance having a binding domain with the required specificity for the secretory structure. Thus, the term covers antibody fragments, derivatives, functional equivalents and homologues of antibodies, including any polypeptide including an immunoglobulin binding domain, whether natural or synthetic. Chimaeric molecules including an immunoglobulin binding domain, or equivalent, fused to another polypeptide are therefore included.
It is also possible that the PSG and/or EB and/or DCB and/or CA structures include nucleic acid molecules. In this case, as would be readily understood by those skilled in the art, the presence of these structures or their contents in tissue could be detected using oligonucleotide probes. The oligonucleotides could also be used in the amplification of the nucleic acid contents, for example, by PCR. The present invention also provides such oligonucleotides.
In an eleventh aspect the present invention provides a method of analysing tissue for pathology, the method comprising detecting the presence of secretory granules in a sample of the tissue.
In a preferred embodiment of the eleventh aspect, the tissue sample is reproductive tissue. Preferably, the reproductive tissue is prostatic, testes, fallopian tube or oviduct tissue. More preferably, the tissue is prostatic tissue.
In a further preferred embodiment of the eleventh aspect the pathology is prostate cancer or prostatitis.
In a further preferred embodiment of the eleventh aspect the analysis involves fixing the tissue sample with a composition according to the third aspect.
In a twelfth aspect the present invention provides a method of diagnosing prostate pathology in a subject which method comprises analysing a fluid sample from the subject for the contents of PSG and/or EB and/or DCB and/or CA structures, or the contents thereof. It will be appreciated that a decrease in any one of the structures of the contents thereof (when compared to a fluid sample from a subject without prostate disease) in the fluid sample is indicative of prostate disease. The body fluid may be blood, serum, semen or urine.
In a preferred embodiment of the twelfth aspect, the disease is prostate cancer or prostatitis.
In a further preferred embodiment of the twelfth aspect the analysis involves immunoanalysis using a binding ligand, preferably an antibody, according to the tenth aspect of the present invention.
In a further preferred embodiment of the twelfth aspect, the contents of any one or more of the structures comprises a sulphate-associated compound according to the eighth or ninth aspects.
In a thirteenth aspect, the present invention provides a method of monitoring the effectiveness of the use an anticancer agent in the treatment of prostate cancer in a subject which method comprises obtaining sequential samples of fluid from the subject over a period of time of treatment and detecting the levels of PSG and/or EB and/or DCB and/or CA structures, or the contents thereof, in the sequential samples.
In a preferred embodiment of the thirteenth aspect of the present invention, the fluid sample is derived from blood serum, seminal fluid, or urine.
It is known that a number of anticancer agents act by inhibiting the assembly of microtubules in tumour cells (13). It now also appears that inhibition of microtubule assembly disturbs secretion of granules, such as prolactin granules, from secretory glands (14). Accordingly, anticancer agents may inhibit the secretion of PSGs from prostate tumour cells.
Accordingly, in a fourteenth aspect the present invention provides a method of screening an agent for anticancer activity, which method comprises
(i) exposing a sample of prostate tumour cells to the agent, and
(ii) monitoring the cells over time for the presence of PSG and/or EB and/or DCB and/or CA structures, wherein the presence of one or more of the structures in the cells indicates that the agent has anticancer activity or allows maturation of cancer cells, thereby making them susceptible to other agents.
In a preferred embodiment, the prostate tumour cells are cultured cells. In a further preferred embodiment, the cultured cells are obtained by transformation of normal prostate luminal cells.
In a fifteenth aspect the present invention provides a method of screening an agent for anticancer activity, which method comprises
(i) exposing a sample of prostate cells to a transforming substance, wherein the level of exposure is sufficient to transform the prostate cells into prostate tumour cells,
(ii) exposing the cells to the agent and
(ii) monitoring the cells over a period of time for the presence of PSG and/or EB and/or DCB and/or CA structures, wherein the maintenance of one or more of the structures in the cells over the period of time, or an increase in the level of one or more of the structures over the period of time, indicates that the agent has anticancer activity.
The prostate cells may be exposed to the agent simultaneously with the transforming substance, or subsequent to exposure to the transforming substance.
The transforming substance may be any substance which transforms normal cells to tumour cells. Examples of suitable transforming substances comprise the Epstein Barr Virus.
In a further preferred embodiment, the cells are monitored for a period of 7 days, more preferably 28 days.
Throughout this specification, the word xe2x80x9ccomprisexe2x80x9d, or variations such as xe2x80x9ccomprisesxe2x80x9d or xe2x80x9ccomprisingxe2x80x9d, will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.